The tale of proteolysis targeting chimeras (PROTACs) for Leucine-Rich Repeat Kinase 2 (LRRK2)

Article abstract of DOI:10.1002/cmdc.202000872

Here we present the rational design and synthetic methodologies towards proteolysis-targeting chimeras (PROTACs) for the recently-emerged target leucine-rich repeat kinase 2 (LRRK2). Two highly potent, selective, brain-penetrating kinase inhibitors were selected, and their structure was appropriately modified to assemble a cereblon-targeting PROTAC. Biological data show strong kinase inhibition and the ability of the synthesized compounds to enter the cells. However, data regarding the degradation of the target protein are inconclusive. The reasons for the inefficient degradation of the target are further discussed.

Full text of DOI:10.1002/cmdc.202000872

Accepted Article
Title: The tale of proteolysis targeting chimeras (PROTACs) for
leucine-rich repeat kinase 2 (LRRK2)
Authors: Alexander Doemling, Markella Konstantinidou, Asmaa Oun,
Pragya Pathak, Bidong Zhang, Zefeng Wang, Frans ter
Brake, Amalia Dolga, and Arjan Kortholt
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000872
Link to VoR: https://doi.org/10.1002/cmdc.202000872
01/2020

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
The tale of proteolysis targeting chimeras (PROTACs) for leucine-
rich repeat kinase 2 (LRRK2)
Markella Konstantinidou,[a]# Asmaa Oun,[b] # Pragya Pathak,[c] # Bidong Zhang,[a] Zefeng Wang,[a] Frans
ter Brake,[a] Amalia Dolga,[b] Arjan Kortholt,[c],[d] and Alexander Dömling [a]*
[a]
Dr. M. Konstantinidou, B. Zhang, Z. Wang, F. ter Brake, Prof. Dr. A. Dömling
Department of Pharmacy, Group of Drug Design
University of Groningen
A. Deusinglaan 1, 9713 AV, Groningen, the Netherlands
E-mail: a.s.s.domling@rug.nl, website: http://www.drugdesign.nl/
[b]
[c]
A. Oun, Dr. A. Dolga
Department of Molecular Pharmacology, Groningen Research Institute of Pharmacy, Behavioral and Cognitive Neurosciences (BCN)
University of Groningen
Groningen, the Netherlands
P. Pathak, Dr. A. Kortholt
Department of Cell Biochemistry, Groningen Institute of Biomolecular Sciences & Biotechnology
University of Groningen
Groningen, the Netherlands
[d]
#
YETEM – Innovative Technologies Application and Research Centre Suleyman Demirel University
Isparta, Turkey
Authors contributed equally.
Supporting information for this article is given via a link at the end of the document.
Abstract: Here we present the rational design and the synthetic
methodologies towards proteolysis targeting chimeras (PROTACs)
for the recently-emerged Parkinson’s target leucine-rich repeat
efforts to develop inhibitors for LRRK2, focusing on ATP
competitive active site kinase inhibitors in particular.
kinase
2 (LRRK2). Two highly potent, selective and brain-
penetrating kinase inhibitors were selected and their structure was
appropriately modified to assemble a cereblon-targeting-PROTAC.
Biological data show strong kinase inhibition and the ability of the
synthesized compounds to enter the cells. However, data regarding
the degradation of the target protein are inconclusive. The reasons
for the inefficient degradation of the target are further discussed.
Parkinson’s disease (PD) is one of the most common
neurodegenerative diseases and although most PD cases are
idiopathic and the etiology is largely unknown, environmental
and genetic factors are also implicated. Among the implicated
genes is the leucine-rich repeat kinase 2 encoding gene
(LRRK2) encoded by PARK8. LRRK2 mutations have been
observed in a number of idiopathic late-onset Parkinson’s
patients and are the most common cause of familial PD.[1-3]
Importantly, recently it has been found that LRRK2 activity was
enhanced in postmortem brain tissue from patients with
idiopathic PD.[4] Therefore, LRRK2 is considered as an essential
player in PD pathogenesis and targeting LRRK2 thus may be
beneficial for PD in general.
Figure 1. LRRK2, a PD target. Above: Functional LRRK2
moieties. Middle:
2D structures of two potent small molecules (PF-06447475, GNE-7915)
inhibiting the LRKK2 kinase. Below left: the crystal structure of PF-06447475
(cyan sticks) with MST3 (grey surface) [PDB 4U8Z], below right overlap of a
docking pose of GNE-7915 (magenta sticks) over PF-06447475 (cyan sticks)
in MST3 active site (grey surface).
LRRK2 is a large protein, consisting of 2527 amino acids and
including multiple domains (Figure 1). Interestingly, it contains
both a kinase and a GTPase domain. Regarding the mutations,
they mostly occur in the GTPase and the kinase domain, leading
Starting from 2006, three patent reviews have been published,
covering the numerous potent scaffolds against the target [7-9]. In
2018, the first clinical trial (NCT03710707) was announced by
Denali Therapeutics Inc., followed by a second clinical trial in
2019 (NCT04056689).[10] The structures of DNL201 and
DNL151 are not yet disclosed.
to increased kinase activity and autophosphorylation.
A
significant number of disease-associated LRRK2 mutations has
been identified to date, among which five missense mutations
(R1441C, R1441G, Y1699C, G2019S and I2020T) are linked to
PD pathogenesis.[5,6] There have been extensive drug discovery
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
[11]
Here we explore the possibility of degrading LRRK2 using a
proteolysis targeting chimera (PROTAC) strategy, instead of
inhibiting its activity. PROTACs in the last few years have shown
tremendous opportunities for modulating challenging or
traditionally considered “undruggable” targets. Despite the fact
that kinase inhibitors for LRRK2 have yet failed to reach the
market, the potential of degrading LRRK2, as an alternative aim,
has not been thoroughly investigated. In our approach, the
starting points for designing and synthesizing PROTACs were
known kinase inhibitors. Due to the abundance of scaffolds in
the literature the following requirements were considered
significant in the choice of the inhibitors: (1) High potency,
preferably low nanomolar inhibitors; (2) High selectivity in the
kinome; (3) Penetration of the blood-brain barrier; (4) Availability
of structural data regarding the binding mode; (5) Solvent
exposed functional group to attach the linker without
compromising the kinase binding; (6) Number of synthetic steps;
(7) Cost and availability of starting materials.
kinase is reported (PDB 4U8Z)
GNE-7915 is also highly
potent (Ki 2 nM in the biochemical assay, IC50 9nM in the cellular
assay), brain penetrant and with high kinome selectivity (1 out of
187).[12] In this case only a docking pose is published with JAK2.
However, the described extensive SAR is sufficient to guide the
structural modifications for a PROTAC.
In particular, the available structural data for PF-06447475 show
that the oxygen of the morpholine participates in a hydrogen
bond that is important for selectivity among the kinome,
however, position 3 of the morpholine is solvent exposed and it
could be used to attach a linker. On the contrary for GNE-7915,
the morpholine moiety seems to be completely solvent exposed
and the hypothesis is that it is not a strict requirement for
binding. In order to choose which E3 ligase to target, the
expression levels in different tissues were checked in the
[13]
database of protein atlas.
A comparative analysis revealed
that cereblon and MDM2 are expressed in the same parts of the
brain, whereas VHL is not. MDM2 is expressed in all tissue in
high levels, so to address possible selectivity issues cereblon
seemed a better choice to begin with. The original routes for
resynthesizing PF-06447475 and GNE-7915 were followed and
then small modifications were considered in order to improve
the yields of intermediates and thus facilitate the synthesis of
PROTACs (Scheme 1).
Based on those requirements, two scaffolds were selected: PF-
06447475, developed by Pfizer and GNE-7915, developed by
Genentech (Figure 1). In particular PF-06447475, has an IC50 of
3 nM in the enzyme assay, 25 nM in the whole cell assay, is
brain penetrant, highly selective in the kinome and does not
show toxicity in rat models. The co-crystal structure with MST3
Scheme 1. Synthetic routes to reach main intermediate 3 for PF-06447475-based PROTACs, upper (original), middle (modified) and transformations to
intermediates with functional groups (below). Reagents and conditions: a) NaH, SEM-Cl, THF, 0oC to rt, 3h, 40% yield, b) (3-cyanophenyl)boronic acd, 1%
Pd(dppf)Cl2, K2CO3, DME – H2O, reflux 3h, 30% yield, c) TFA, rt, 24h, 60% yield, d) trityl chloride, CHCl3, Et3N, rt, 1h, quantitative, e) (3-cyanophenyl)boronic acd,
0.004% Pd(dppf)Cl2, NaHCO3, toluene – EtOH, reflux 24h, 60% yield, f) TFA, DCM, rt, 24h, 90% yield, g) morpholine for (6) tert-butanol, DIPEA, reflux 3h, or ethyl
morpholine-2- carboxylate for (7) or tert-butyl N-(morpholin-2-ylmethyl)carbamate for (9) or N-Boc-piperazine for (10), EtOH, DIPEA, MW, 150oC, 1h, yields 26 –
30%, h) LiOH, THF – H2O, 65% yield.
For PF-06447475, the main intermediate (3) was synthesized
in 3 steps as shown in scheme 1. The original route
(upper route) led to intermediate (3) with only 8% overall yield
and required two column purifications. However, by simply
changing the protecting group in the first step from (2-
substitution was performed under microwave irradiation
[11,14]
instead of reflux.
For GNE-7915 the original route
coupling with morpholine and
[12]
includes an amide
nucleophilic aromatic
a
substitution, with a starting material that also needs to be
synthesized. In GNE-7915 the morpholine part is solvent
exposed and is also the position where the linkers can be
attached. For the benefit of the overall route it is best to
introduce the morpholine at the end of the synthesis. We
developed an alternative route, starting from the commercially
available 4-amino-2-fluoro-5-methoxybenzoic acid (Scheme
2). The initial substitution of the fluorine with a methoxy
group, was followed by an esterification and a reduction of
the nitro group to receive intermediate (14). The next step
was a nucleophilic aromatic substitution using the bifunctional
chloromethoxyethyl)trimethylsilane
(SEM-Cl)
to
trityl-
chloride[15]
,
the yield became almost quantitative.
Optimization of the Suzuki coupling also increased the yield
significantly, and now the optimized route (middle route), led
to intermediate (3) with 54% yield over three steps, requiring
only one column purification. After obtaining intermediate (3),
the original inhibitor PF-06447475 (6) was synthesized with a
nucleophilic aromatic substitution with morpholine. In order to
attach suitable linkers for the PROTACs, morpholines
substituted on position 3 were used, bearing either an ester
group or a Boc-protected amine to obtain intermediates (7)
and (9) respectively. Boc-piperazine was also used in a
similar way for intermediate (10). The nucleophilic aromatic
building
block
2,4-dichloro-5-(trifluoromethyl)pyrimidine.
Interestingly, the substitution can be performed selectively by
using zinc chloride as catalyst.[16] The obtained intermediate
(15), undergoes a substitution on the second chloride (16),
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
followed by a hydrolysis to give the carboxylic acid (17),
which is the key intermediate in this synthesis. Overall, the
yield is 32% over 6 steps with only one column purification.
The carboxylic acid (17) was used in amide coupling
reactions with morpholine to obtain GNE-7915 (11) and also
in amide couplings directly with CRBN building blocks or with
a substituted morpholine to obtain the amine intermediate
(18).
Scheme 2. Synthetic routes to GNE-7915: upper (original), middle (modified) and intermediates for GNE-7915-based PROTACs (below). Reagents and
conditions: a) MeOH, KOH, rt, 2h, yield 92%, b) EEDQ, EtOH, reflux 5h, quantitative, c) stannous chloride, EtOH – H2O, reflux 4h, quantitative, d) 2,4-dichloro-5-
(trifluoromethyl)pyrimidine, zinc chloride, diethylether, DCM, tert-butanol, triethylamine, 0oC to rt, 48 h, 40% yield, e) ethanamine, triethylamine, THF, 0oC to rt, 1h,
88% yield, f) LiOH, THF – H2O, 97% yield, g) morpholine for (11) or tert-butyl N-(morpholin-2-ylmethyl)carbamate for (18), EEDQ, CHCl3, reflux 2h, 40% yield.
Scheme 3. Synthetic routes for CRBN-building blocks. Reagents and conditions: a) sodium acetate, acetic acid, overnight reflux, yield 90%, b) H2, Pd/C, rt, 24h,
yield 92%, c) potassium acetate, acetic acid, reflux 3h, yield 32%, d), THF, reflux, 30min, yield 92%, e) sodium iodide, potassium carbonate, THF, rt, yield 81%, f)
sodium acetate, acetic acid, overnight reflux, yield 85%, g) N-Boc-1,3-propanediamine or N-Boc-1,4-butanediamine, DIPEA, DMF, overnight reflux, yield 60%.
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
For the synthesis of cereblon building blocks two starting
materials were used: the 4-nitroisobenzofuran-1,3-dione and
the 4-fluoroisobenzofuran-1,3-dione (Scheme 3). The first
synthetic step is the condensation of 4-nitroisobenzofuran-
1,3-dione with 3-aminopiperidine-2,6-dione to obtain the nitro-
substituted imide (19), which is then reduced to the aniline
group to obtain pomalidomide (20). Pomalidomide (20) was
then used in an anhydride opening reactions to obtain the
carboxylic acid (21). Pomalidomide (20) was also used in an
acylation reaction with 2-chloroacetyl chloride to obtain the
intermediate (22), which with a substitution reaction led to
intermediate (23). In a similar way, the condensation of 4-
fluoroisobenzofuran-1,3-dione with 3-aminopiperidine-2,6-
dione was performed to obtain the fluoro-substituted imide
(24), which underwent a nucleophilic aromatic substitution
with linear Boc-protected diamines to obtain intermediates
(25) and (26).
After synthesizing the appropriate kinase intermediates and
the cereblon building blocks, the final PROTAC compounds
were synthesized with amide coupling reactions. In all the
cases of Boc-protected intermediates, the deprotection was
performed with HCl in dioxane and the obtained salts were
used directly in the amide coupling without purification. An
overview of structures and coupling yields is shown in
scheme
4.
Scheme 4. Structures of PF-06447475-based-CRBN-PROTACs and GNE-7915-based-CRBN-PROTACs.
Then, the four PF-06447475/CRBN-based PROTACs and the
three GNE-7915/CRBN-based PROTACs, as well as the
original resynthesized inhibitors were biologically evaluated.
cells and attach to the kinase pocket of LRRK2, (Figure 2A).
We next determined the ability of different PROTACs to
degrade LRRK2 in LRRK2 parental RAW 264.7 cells. For the
The
original inhibitors were used as reference compounds.
initial screening the cells were incubated with
2
First, an in vitro kinase assay with the phosphorylation rate of
a key LRRK2 autophosphorylation site (S1292-LRRK2) as
readout, revealed that the compounds were indeed potent
kinase inhibitors (Figures S1 and S2). Then, in order to test
whether the PROTACs were cell-permeable and able to bind
to the kinase pocket of LRRK2, confocal microscopy was
performed. It is known that incubation of LRRK2 with ATP
concentrations (1 and 10 µM) of PROTACs for 24, 48 and 72
hours. Data from western blotting did not show any significant
changes in the LRRK2 protein levels between the PROTAC
treated cells and cells treated with the original kinase
inhibitors, indicating that PROTACs were not able to cause
LRRK2 degradation (Figures 2B and 2C). Furthermore,
ubiquitination assays showed that one of the most potent and
cell permeable PROTACs, PROTAC (33) (IC50 = 14.69 ± 6.14
nM) was unable to increase the ubiquitin signal compared to
the DMSO or original inhibitor compound (11) (IC50 = 17.3 ±
6.76nM) (Figure 2D).
competitive
inhibitors
results
in
relocalization
of
overexpressed GFP-tagged LRRK2 onto microtubules.[17-20]
HEK cells transfected with GFP-tagged LRRK2 were
incubated with different PROTACs along with the original
kinase inhibitors and DMSO as controls. Formation of green
filaments was observed after incubation with the original
inhibitors and most of the PROTACs conferring the ability of
the majority of the synthesized PROTACs to penetrate the
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
Figure 2. PROTACs are cell-permeable yet not increasing LRRK2 degradation and ubiquitination. (A) Confocal microscopy of GFP tagged LRRK2
transfected HEK293 cells showing LRRK2 localization inside cells treated with DMSO, original kinase inhibitor and one of the PROTACs. The images show that
the original kinase inhibitor and the PROTAC compound are cell-permeable and able to induce LRRK2 localization to the microtubules depicted as green
filaments. (B) and (C), representative western blots of LRRK2 parental RAW 264.7 cells treated with 10 µM of different PROTACs together with the original kinase
inhibitors and DMSO controls for 24 hours and immunoblotted with LRRK2 and GAPDH as a loading control. The quantification of the blots (n=3) shows that
different PROTACs are not affecting LRRK2 degradation compared to the original kinase inhibitor. (D) GFP tagged LRRK2 transfected HEK293 cells were treated
with DMSO, original kinase inhibitor (11) and PROTAC (33) with or without MG132 for 24 hours. Ubiquitin assay was performed on the collected cells. The
samples were immunoblotted with anti-ubiquitin (P4D1) for the ubiquitin signal and anti-GFP for the LRRK2 signal. The blot shows that MG132 can enhance the
ubiquitin signal and that neither the original kinase inhibitor (11) nor PROTAC (33) increase LRRK2 ubiquitination compared to the DMSO treated control.
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
Despite the fact that synthesized PROTACs were able to
enter the cells and bind to the target, there a few possible
hypotheses explaining their inability to induce ubiquitination
and degradation of LRRK2. It could be argued at this point
that the synthesized number of PROTACs was limited and
more variations on the linkers, including length, flexibility and
attachment points, could be explored. However, since two
different highly potent LRRK2 ligands failed to induce the
degradation of the target, despite showing target engagement
and cell permeability, there are also other possible
explanations to consider. One plausible hypothesis, is that
due to the observed re-localization of LRRK2 to the
microtubules and formation of stable filaments, LRRK2 might
not be accessible to the E3 ligase component to form a
ternary LRRK2-PROTAC-E3 ligase complex. Moreover, since
the full-length structure of LRRK2 is not known, the proximity
of lysine residues suitable for ubiquitination and degradation
to the kinase site might not be optimal. To date, the full length
structure of LRRK2 is not solved. Recently, a high resolution
cryo-EM structure of the catalytic half of LRRK2 including the
RoC/GTPase, COR, kinase and WD40 domains was
reported.[20] The structure revealed that the kinase and
GTPase domains are in close proximity. Notably, in the
absence of kinase inhibitors, the kinase was in an inactive /
open conformation in the cryoEM structure, whereas data
showed that the microtubule-associated LRRK2 had the
Experimental Section
Experimental Details (supporting information). General
procedures, characterization data (1H-NMR, 13C-NMR),
biological screening (kinase assays, cell culture, microscopy,
western blotting, ubiquitin assay).
Acknowledgements
This research has been supported (to AD) through ITN
“Accelerated Early stage drug dIScovery” (AEGIS, grant
agreement No 675555). Moreover, funding was received by
the National Institute of Health (NIH) (2R01GM097082-05),
the European Lead Factory (IMI) (grant agreement number
115489), the Qatar National Research Foundation (NPRP6-
065-3-012). and, COFUNDs ALERT (grant agreement No
665250) and Prominent (grant agreement No 754425) and
KWF Kankerbestrijding grant (grant agreement No 10504).
A.M.D. is the recipient of a Stichting Parkinson Fonds (SPF)
grant and a Rosalind Franklin Fellowship co-funded by the
European Union and the University of Groningen. AK is
holder of a TUBITAK 2232 scholorship and additional funding
was received from the The Michael J. Fox Foundation for
Parkinson's Research.
kinase domain in
a
closed and potentially active
conformation. Moreover, kinase inhibitors also had an effect
on the kinase domain conformation and in particular,
inhibitors that promoted LRRK2-microtubule binding favored
Conflicts of interest
The authors declare no conflicts of interest.
the closed kinase conformation. The proposed model
[21]
indicates the complexity of targeting LRRK2. Villa et al
,
using cryoEM and integrative modeling, revealed the
structure of LRRK2 in situ and showed that the GTPase
domain is closer to the microtubule interface, in contrast to
the kinase domain which is exposed to the cytoplasm.
ABBREVIATIONS
LRRK2, leucine-rich repeat kinase 2; PROTAC, proteolysis
targeting chimera; CRBN, cereblon; VHL, Von Hippel Lindau;
MDM2, mouse double minute 2 homolog
Another aspect to be taken into account, are the properties of
LRRK2 inhibitors. Although numerous scaffolds have been
reported, in most of the cases, there is lack of structural data
and differences in selectivity. This seems to be affecting also
the degradation potential of LRRK2 inhibitors. At the time of
manuscript submission, a patent highlight indicated the
degradation of LRRK2 and compared two types of inhibitors:
the aminopyrimidine analogs and the indazole analogs.[22-23]In
agreement with our observations, the aminopyrimidine
analogs failed to degrade the target. On the other hand, the
indazole analogs seemed to be able to reduce the levels of
LRRK2. The data taken together with our results, show that
the selection of LRRK2 inhibitors is crucial for the
development of degraders. Additionally, very recently, a high-
throughput screen resulted in the discovery of a small
molecule, which showed remarkable selectivity for G2019S-
LRRK2, the most common LRRK2 pathogenic mutation.[24]
This could be an interesting starting point for future studies of
selective G2019S-LRRK2 degraders.
To date, although PROTACs have been successful in
challenging targets, the task of developing degraders is not
trivial. Especially in the case of LRRK2, which remains an
elusive drug target, a better understanding of its structure and
conformational changes, as reported very recently, is
necessary in order to further explore the possibility of
degrading it and additionally to understand the reasons for
the observed differences when highly potent LRRK2 inhibitors
were modified into potential degraders. Overall, in this work,
we aim to underline the challenges in degrading a target, for
which the full structure is not yet known. We believe that
future work will enable the rational development of a
successful LRRK2-targeting PROTAC, following a more
complete picture of LRRK2’s structure and dynamics.
Keywords: LRRK2 • Cereblon • PROTAC • degradation •
Parkinson’s disease
[1] W.P. Gilks, P.M. Abou – Sleiman, S. Gandhi, S. Jain, A.
Singleton, A.J. Lees, K. Shaw, K.P. Bhatia, V. Bonifiati, N.P.
Quinn, J. Lynch, D.G. Healy, J.L. Holton, T. Revesz, N.W.
Wood. Lancet 2005, 365(9457), 415-416.
[2] S. Lesage, S. Janin, E. Lohmann, A.L. Leutenegger, L.
Leclere, F. Viallet, P. Pollak, F. Durif, S. Thobois, V. Layet,
M. Vidailhet, Y. Agid, A. Dürr, A. Brice. Arch Neurol. 2007,
64(3), 425-430.
[3] S. Bardien, S. Lesage, A. Brice, J. Carr. Parkinsonism Relat
Disord. 2011, 17(7), 501-508.
[4] R. Di Maio, E.K. Hoffman, E.M. Rocha, M.T. Keeney, L.H.
Sanders, B.R. De Miranda, A. Zharikov, A. Van Laar, A.F.
Stepan, T.A. Lanz, J.K. Kofler, E.A. Burton, D.R. Alessi, T.G.
Hastings, J.T. Greenamyre. Sci. Transl. Med. 2018,
10(451):eaar5429.
[5] B.I. Giasson, V.M. Van Deerlin. Neurosignals 2008, 16, 99–
105.
[6] D.J. Moore. Parkinsonism Relat. Disord. 2008, 14 Suppl 2,
S92-98.
[7] X. Deng, H.G. Choi, S.J. Buhrlage, N.S. Gray. Expert Opin.
Ther. Pat. 2012, 22(12), 1415-1426.
[8] R.R. Kethiri, R. Bakthavatchalam. Expert Opin. Ther. Pat.
2014, 24(7), 745-757.
[9] P. Galatsis. Expert Opin. Ther. Pat. 2017, 27(6), 667-676.
[10] https://clinicaltrials.gov/
[11] J.L. Henderson, B.L. Kormos, M.M. Hayward, K.J. Coffman,
J. Jasti, R.G. Kurumbail, T.T. Wager, P.R. Verhoest, G.S.
Noell, Y. Chen, E. Needle, Z. Berger, S.J. Steyn, C.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
Houle, W.D. Hirst, P. Galatsis . J. Med. Chem. 2015, 58(1),
419-432.
[12] A.A. Estrada, X. Liu, C. Baker-Glenn, A. Beresford, D.J.
Burdick, M. Chambers, B.K. Chan, H. Chen, X. Ding, A.G. Di
Pasquale, S.L. Dominguez, J. Dotsonm, J. Drummond, M.
Flagella, S. Flynn, R. Fuji, A. Gill, J. Gunzer-Toste, S.F.
Harris, T.P. Heffron, T. Kleinheinz, D.W. Lee, C.E. Le
Pichon, J.P. Lyssikatos, A..D Medhurst, J.G. Moffat, S.
Mukund, K. Nash, K. Scearce-Levie, Z. Sheng, D.G. Shore,
T. Tran, N. Trivedi, S. Wang, S. Zhang, X. Zhang, G. Zhao,
H. Zhu, Z.K. Sweeney. J. Med. Chem. 2012, 55(22), 9416-
9433.
[13] https://www.proteinatlas.org/
[14] P. Galatsis, (Pfizer), US2017002000A1, 2017.
[15] Y-L. Chen, (Novartis), WO2010015637A1, 2010.
[16] J.C. Kath, (Pfizer), WO2005023780A1, 2005.
[17] N.Dzamko, M. Deak, F. Hentati, A.D. Reith, A.R. Prescott,
D.R. Alessi, R.J. Nichols. Biochem J. 2010, 430(3),405-413.
[18] X. Deng, N. Dzamko, A. Prescott, P. Davies, Q. Liu, Q.
Yang, J.D. Lee, M.P. Patricelli, T.K. Nomanbhoy, D.R.
Alessi, N.S. Gray. Nat Chem Biol. 2011, 7(4), 203-205.
[19] M.B. Ramírez, A.J. Ordóñez, E. Fdez, J. Madero-Pérez, A.
Gonnelli, M. Drouyer, M.C. Chartier-Harlin, J.M. Taymans, L.
Bubacco, E. Greggio, S. Hilfiker. Hum Mol Genet. 2017,
26(14),2747-2767.
[20] C.K. Deniston, J. Salogiannis, S. Mathea, D.M. Snead, I.
Lahiri, M. Matyszewski, O. Donosa, R. Watanabe, J.
Böhning, A.K. Shiau, S. Knapp, E. Villa, S.L. Reck-Peterson,
A.E. Leschziner. Nature 2020, doi:10.1038/s41586-020-
2673-2.
[21] R. Watanabe, R.L. Buschauer, J. Böhning, M. Audagnotto,
K. Lasker, L. Tsan-Wen, D. Boassa, S. Taylor, E. Villa. Cell
2020, S0092-8674(20)30995-8.
doi:10.1016/j.cell.2020.08.004.
[22] R.B. Kargbo, ACS Med. Chem. Lett. 2020, 11, 2070 – 2071
[23] N.S. Gray, J. Hatcher (Dana-Farber Cancer Institute), WO
2020/081682 A, 2020.
[24] A.W. Garofalo, J. Bright, S. De Lombaert, A.M.A. Toda, K.
Zobel, D. Andreotti, C. Beato, S. Bernardi, F. Budassi, L.
Caberlotto, P. Gao, C. Griffante, X. Liu, L. Mengatto, M.
Migliori, F. M. Sabbatini, A. Sava, E. Serra, P. Vincetti, M.
Zhang, H.J. Carlisle. J. Med. Chem 2020, doi:
10.1021/acs.jmedchem.0c01243.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000872
ChemMedChem
COMMUNICATION
Entry for the Table of Contents
Here, we present our efforts for developing LRRK2-targeting PROTACs, starting from two highly-potent LRRK2 inhibitors. Although
the synthesized PROTACs showed cell permeability and interacted with the target, they failed to induce target degradation. The
present work highlights the challenges of PROTAC design for multidomain protein targets where the structure is not fully known and
the dynamics are not fully understood.
8
This article is protected by copyright. All rights reserved.